Human cytomegalovirus (HCMV) causes latent and persistent infections in 60-90% of the population. Virus proliferation significantly increases the morbidity and/or mortality in immunocompromised individuals such as newborns, organ transplant recipients, AIDS patients, as well as the elderly. HCMV is a significant health challenge requiring the development of a multi-facet approach for the generation of effective and safe treatments to limit HCMV proliferation. The current treatments against HCMV are anti-viral drugs such as ganciclovir, valganciclovir, foscarnet, and cidofovir that target the viral polymerase. A major draw-back to these drugs is their toxicity, drug-drug interactions, and the development of drug resistance strains of the virus upon prolonged treatment. The current application proposes to develop and utilize high-throughput screening assays to identify lead compounds that limit HCMV proliferation and dissemination in response to PA-10-213, 'Development of Assays for High-Throughput screening for use in Probe and Pre-therapeutic Discovery.' Our central hypothesis is that targeting multiple stages of HCMV proliferation would significantly reduce HCMV disease through the effective inhibition of the HCMV life cycle. Thus, we propose to discover compounds that block different steps of HCMV proliferation through the development of high-throughput screening assays. These assays will utilize HCMV variants that express chimeric viral proteins fused to yellow fluorescent proteins or luciferase. In addition, secondary assays to determine the viral and cellular specificity for patient-derived clinical strais of hit compounds and the compound's general mode of action are routine protocols in the lab. The development of robust high-throughput screens for the identification of anti-HCMV drugs is an essential first step to generating effective therapies against HCMV and the associated diseases. Potential lead compounds may target various steps of the HCMV life cycle such as viral entry, viral protein expression, viral egress, and virus particle release from the infected cll. These compounds in combination with current therapy as a multi-drug cocktail would likely be very effective at treating patients with a high HCMV viral load and limit HCMV associated diseases.